Patri Vergara

Glucagonlike Peptide-1 (GLP-1) Participation in Ileal Brake Induced by Intraluminal Peptones in Rat

The aim of this study was to determine themechanisms implicated in the gastrointestinal inhibitioninduced by ovoalbumin hydrolysate infusedintraluminally. We studied the site of action, thepossible implication of GLP-1, and the nervousmechanisms involved. We prepared anesthetizedSprague-Dawley rats with strain gauges in the antrum,duodenum, and proximal jejunum and a catheter in theduodenum or ileum for peptone infusion. Both intraduodenal(N = 6) and intraileal (N = 5) infusion of ovoalbuminhydrolysate induced inhibition of spontaneous motoractivity in the antrum, duodenum, and proximal jejunum. Duodenal inhibition induced by intraduodenal (N= 6) or intraileal (N = 6) infusion of ovoalbuminhydrolysate was reversed by intraarterial infusion ofGLP-1 receptor antagonist, exendin (9-39) (3 ×10-8 mol/kg/40 min). Finally, a combination of theadrenergic blockers phentolamine and propranolol (1mg/kg, each; N = 7) completely blocked inhibitorygastrointestinal motor actions caused by intraduodenal infusion of ovoalbumin hydrolysate. This studydemonstrates that peptone, intraluminally infused,participates in the regulation of gastrointestinalmotility through stimulation of adrenergic pathways in anaesthetized rats. Moreover, these effects arepartly mediated by GLP-1 secretion. The ileum seems tobe the site of action, indicating a role of GLP-1 on theileal break mechanism.

Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation.

Background: Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. Methods: Indomethacin administration to rats induced a long‐lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short‐circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. Results: Rats with induced enteritis in both phases demonstrated a long‐lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. Conclusions: The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.

Mast cell stabilizer ketotifen [4-(1-methyl-4-piperidylidene)-4h-benzo[4,5]cyclohepta[1,2-b]thiophen-10(9H)-one fumarate] prevents mucosal mast cell hyperplasia and intestinal dysmotility in experimental Trichinella spiralis inflammation in the rat.

Trichinella spiralis infection in rats induces hypermotility and an abnormal response to cholecystokinin (CCK) similar to motor disturbances observed in irritable bowel syndrome. Mast cell hyperplasia is also characteristic of this experimental model. The aim of our study was to correlate mast cell activity with the development of dysmotility and to demonstrate whether the mast cell stabilizer ketotifen [4-(1-methyl-4-piperidylidene)-4H-benzo[4,5]cyclohepta[1,2-b]thiophen-10(9H)-one fumarate] could prevent the development of intestine hypermotility. Sprague-Dawley rats were infected with T. spiralis and, 5 days after infection, treated with the mast-cell stabilizer ketotifen (10 mg/kg/day). Twelve days after infection, intestinal spontaneous motor activity and response to CCK were evaluated by means of strain-gauge transducers. Immunohistochemistry for rat mast cell protease II (RMCPII), cyclooxygenase (COX)-2, and inducible nitric-oxide synthase (iNOS) was performed in intestinal specimens. In addition, RMCPII and myeloperoxidase were determined in serum. Infected control rats showed hypermotility, mast cell hyperplasia, increased RMCPII levels, increased myeloperoxidase, and overexpression of COX-2 and iNOS. In contrast, ketotifen-treated rats showed spontaneous intestinal motility and CCK response similar to the noninfected control rats. Mast cell hyperplasia and RMCPII were reduced in ketotifen-treated rats. Inflammatory parameters were less modified by ketotifen, but those animals that received the longest ketotifen treatment showed a slight amelioration in these parameters. These results indicate that mast cells are implicated in the development of hypermotility. The treatment with ketotifen prevented hypermotility and mast cell hyperplasia and diminished mucosal mast cell activity.

Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice

Stress leads to altered gastrointestinal neuro‐immune responses. We characterized the interaction between stress and gut commensal microbiota and their role modulating colonic responses to stress, the induction of inflammation, the expression of sensory‐related markers, and visceral sensitivity.

Heterogeneity in electrical activity of the canine ileal circular muscle: interaction of two pacemakers.

Abstract In cross‐sectioned slabs from the muscularis externa or in the isolated circular muscle devoid of longitudinal muscle and myenteric plexus (MyP) of ileum, a slow wave, sigmoidal or triangular in shape, was recorded from microelectrode impalements near the deep muscular plexus (DMP) region in the whole‐thickness preparation. From the MyP region of whole‐thickness preparations, a slow wave which oscillated at a similar frequency (9–10 cycles min−1) was characterized by a fast upstroke and a square shape. Slow waves of μmixed pattern were recorded in the outer circular muscle (OCM) while triangular slow waves were present near the submucous plexus (SMP). In this preparation but not in isolated circular muscle, the inhibitory junction potentials (IJPs) produced by supramaximal electrical field stimulation triggered slow waves. The amplitudes (15–25 mV) of spontaneous and triggered slow waves (TSWs) were greatest in the MyP region, significantly so compared to those of DMP and SMP regions and to those in all regions of isolated circular muscle. Frequencies of slow waves recorded from the MyP and DMP were slightly but significantly higher than those recorded from either the OCM or the SMP or from all regions of isolated circular muscle. A 10–15 mV gradient in resting membrane potential (more hyperpolarized near MyP) existed across the intact (but not the isolated) circular muscle layer. Both types of slow waves, TSWs and IJPs were unaffected by muscarinic, adrenergic or tachykinergic blockade. We suggest that a MyP pacemaker network generated a plateau‐type slow wave while a DMP one induced a triangular slow wave. Each source can function independently but the MyP network may dominate and entrain DMP slow waves.

Mucosal mast cells are involved in CCK disruption of MMC in the rat intestine

Our aim was to determine if mucosal mast cells could be activated by endogenous CCK and, as a consequence, mediate CCK actions in the small intestine. Rats were prepared for electromyography to record electrical activity in the small intestine. In another group of animals, the duodenum was perfused to measure rat mast cell protease II (RMCP II) as indicative of mast cell degranulation. Endogenous CCK release was induced by administration of soybean trypsin inhibitor (SBTI) in conscious rats or by intraduodenal perfusion of ovalbumin hydrolysate (OVH) in anesthetized rats. CCK concentration was measured by bioassay on pancreatic acini. SBTI in control rats disrupted migrating motor complexes (MMC) for >40 min. In rats treated with the mast cell stabilizer ketotifen, SBTI did not induce any change in the MMC pattern. RMCP II concentration in the duodenal perfusate significantly increased after OVH. Perfusate from ketotifen-treated animals did not show any significant increase in RMCP II values during OVH perfusion, although CCK plasma concentration was not different from the control group. Furthermore, infusion of the CCK-B receptor antagonist L-365,260 significantly blocked the increase of RMCP II concentration after OVH. Our results indicate that mucosal mast cells are degranulated by endogenous CCK release through stimulation of CCK-B receptors. Therefore mucosal mast cells participate in CCK intestinal actions.

Influence of nitric oxide and vasoactive intestinal peptide on the spontaneous and triggered electrical and mechanical activities of the canine ileum

Modulation of canine ileal pacemaker activity by nitric oxide (NO) or vasoactive intestinal peptide (VIP) was studied during recording of the intracellular electrical and mechanical activity from the entire muscularis externa and from an isolated circular muscle preparation both cut in the long axis of the circular muscle. In the whole-thickness preparation with cholinergic and adrenergic nerve function blocked, the inhibitory junction potentials (IJPs) recorded near the myenteric plexus (MyP) or deep muscular plexus (DMP) were abolished by omega-conotoxin GVIA (omega-CTX, 10(-7) to 3 x 10(-7) M), tetrodotoxin (TTX, 1 microM), or the NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA at 50 microM). IJPs from electrical field stimulation triggered slow waves (TSWs); after TTX or omega-CTX, TSWs still occurred, advanced in time and increased in amplitude after TTX. Addition of L-NNA advanced the onset of the TSWs after omega-CTX. TTX, L-NNA, or omega-CTX left the resting membrane potentials, the characteristics of spontaneous slow waves, or TSWs evoked by a long stimulating pulse unchanged. L-NNA at 100 microM enhanced the amplitude but not the frequency of spontaneous slow waves. TTX and NOS blockers all increased circular muscle contractions associated with the spontaneous slow waves and TSWs. In isolated circular muscle preparations, the NOS inhibitors N omega-nitro-L-arginine methyl ester (L-NAME at 300 microM) or L-NNA at 100 microM abolished the IJPs and increased the regularity and amplitude of spontaneous slow waves and associated contractions, but TSWs could not be evoked before or after NOS inhibition. The NO donor 3-morpholinosydnonimine hydrochloride (SIN-1) at 200 microM caused hyperpolarizations (10-15 mV) similar to the IJP mediator, attenuated the IJPs, and abolished mechanical activities. SIN-1 increased the slow wave frequency but decreased the amplitude and duration of spontaneous slow waves and TSWs. VIP (10(-6) M) decreased contraction and slow wave amplitude and prolonged IJP duration without affecting membrane potential or slow wave frequency. We conclude that spontaneous slow waves and TSWs originate independently of neural activity. Pacemaking regions possess inhibitory neural inputs that release NO to mediate IJPs and relaxation and influence the delay before a TSW. NO (not VIP) release from nerves inhibits initiation of spontaneous slow waves or TSWs near the MyP, and spontaneous NO release modulates pacemaking activity from the DMP.

Hypersensitivity to ovalbumin induces chronic intestinal dysmotility and increases the number of intestinal mast cells.

Abstract  Undiagnosed food allergies have been proposed as possible causes of promoting and perpetuating irritable bowel syndrome . Our aim was to find out if sensitization could induce chronic functional motor disturbances in the intestine and the mechanisms implicated. Rats were sensitized to ovalbumin (OVA) following three hypersensitivity induction protocols, two parenteral and one oral. Rat mast cell protease II (RMCP II) release in response to OVA challenge and immunoglobulin E (IgE) concentration were measured in serum. At least 1 week after challenge, small intestinal motility was evaluated using strain gauges. Intestinal tissue samples from orally sensitized rats were checked for in vitro stimulation with OVA. Mucosal mast cells were counted from duodenum sections. All sensitized rats showed intestinal hypermotility. Only rats sensitized by parenteral procedure showed an increase in RMCP II after OVA challenge in serum. IgEs increased only in the Bordetella pertussis sensitized group. Small intestine sections from orally sensitized rats released more RMCP II than sections from control rats. All sensitized rats showed an increase in the number of mucosal mast cells in duodenum. In conclusion, hypersensitivity to food proteins induces chronic motor alteration that persists long after antigen challenge and an excited/activated state of sensitized mucosal mast cells.

Sympathetic pathways mediate GLP-1 actions in the gastrointestinal tract of the rat

The aim of this study was to establish the actions of GLP-1 (7-37) on gastrointestinal motility in rats. We prepared anaesthetized Sprague-Dawley rats with strain-gauges in the antrum, duodenum and the proximal jejunum and a catheter in the aorta close to the coeliac artery for close infusion of substances. Intraarterial GLP-1 infusions (3 x 10(-10) and 3 x 10(-9) moles/kg per 10 min) (n = 8) induced inhibition of spontaneous motor activity in the antrum, duodenum and proximal jejunum. Inhibition induced by GLP-1 was reversed by i.v. infusion of GLP-1 receptor antagonist, Exendin (9-39) (3 x 10(-8) moles/kg per 10 min) (n = 6). Neither the presence of L-NNA (10(-5) moles/kg) (n = 9) nor the VIP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP (3 x 10(-8) moles/kg per 10 min) (n = 8) modified responses to GLP-1. However, a combination of the adrenergic blockers phentolamine and propranolol (1 mg/kg each) (n = 8) completely blocked motor actions of GLP-1 in all the organs studied. Moreover, inhibition of gastrointestinal motor activity by GLP-1 was blocked by previous infusion of hexamethonium (10 mg/kg) (n = 4). This study demonstrates that GLP-1 inhibits gastrointestinal motor activity of the rat acting on specific GLP-1 receptors and via stimulation of adrenergic pathways.

Evidence supporting a role for ATP as non-adrenergic noncholinergic inhibitory transmitter in the porcine ileum

The aim of this study was to investigate the nature of the non-adrenergic non-cholinergic (NANC) inhibitory transmitter of the circular muscle of the porcine ileum. For this purpose, the effects of putative NANC mediators i.e. NO, vasoactive intestinal polypeptide (VIP) and ATP were measured in isolated organ bath experiments (in basal conditions and after incubation with neostigmine 3 x 10[-5] M) and using the microelectrode technique. The NO donor sodium nitroprusside (NaNP) up to 10(-4) M, VIP up to 10(-7) M and ATP up to 10(-4) M failed to cause significant relaxation in the basal state. However, all of them induced marked relaxations when the tissue had been preincubated with neostigmine (3 x 10[-5] M) which was added to increase basal mechanical activity. The resting membrane potential (RMP) was unaffected by NaNP(up to 10(-4) M and VIP up to 10(-7) M whereas ATP (up to 10[-4] M) induced a transient hyperpolarization. The inhibitory junction potentials (IJPs) induced by electrical field stimulation (EFS) were not affected by N omega-nitro-L-arginine (L-NNA) (10[-4] M) whereas suramin, a purinoceptor antagonist, decreased (10[-4] M) or abolished (10[-3] M) the IJPs. Relaxations induced by ATP in neostigmine preincubated tissue were resistant to 10(-6) M tetrodotoxin, an axonal blocker, and inhibited by suramin. Apamin (10[-6] M, a small conductance calcium activated potassium channel blocker, completely abolished the IJP (n=5) and significantly decreased the relaxation induced by ATP (n=5). The present data provide support to the hypothesis that ATP is the NANC inhibitory transmitter in the porcine ileum acting on P2 muscular receptors. Nevertheless, VIP and NaNP do also cause relaxation of preparations preincubated with neostigmine.